The Multistage Antimalarial Compound Calxinin Perturbates P. falciparum Ca2+ Homeostasis by Targeting a Unique Ion Channel.

Malaria elimination urgently needs novel antimalarial therapies that transcend resistance, toxicity, and high costs. Our multicentric international collaborative team focuses on developing multistage antimalarials that exhibit novel mechanisms of action. Here, we describe the design, synthesis, and evaluation of a novel multistage antimalarial compound, 'Calxinin'. A compound that consists of hydroxyethylamine (HEA) and trifluoromethyl-benzyl-piperazine. Calxinin exhibits potent inhibitory activity in the nanomolar range against the asexual blood stages of drug-sensitive (3D7), multidrug-resistant (Dd2), artemisinin-resistant (IPC4912), and fresh Kenyan field isolated Plasmodium falciparum strains. Calxinin treatment resulted in diminished maturation of parasite sexual precursor cells (gametocytes) accompanied by distorted parasite morphology. Further, in vitro liver-stage testing with a mouse model showed reduced parasite load at an IC50 of 79 nM. A single dose (10 mg/kg) of Calxinin resulted in a 30% reduction in parasitemia in mice infected with a chloroquine-resistant strain of the rodent parasite P. berghei. The ex vivo ookinete inhibitory concentration within mosquito gut IC50 was 150 nM. Cellular in vitro toxicity assays in the primary and immortalized human cell lines did not show cytotoxicity. A computational protein target identification pipeline identified a putative P. falciparum membrane protein (Pf3D7_1313500) involved in parasite calcium (Ca2+) homeostasis as a potential Calxinin target. This highly conserved protein is related to the family of transient receptor potential cation channels (TRP-ML). Target validation experiments showed that exposure of parasitized RBCs (pRBCs) to Calxinin induces a rapid release of intracellular Ca2+ from pRBCs; leaving de-calcinated parasites trapped in RBCs. Overall, we demonstrated that Calxinin is a promising antimalarial lead compound with a novel mechanism of action and with potential therapeutic, prophylactic, and transmission-blocking properties against parasites resistant to current antimalarials.

The emerging paradigm of calcium homeostasis as a new therapeutic target for protozoan parasites.

Calcium channels (CCs), a group of ubiquitously expressed membrane proteins, are involved in many pathophysiological processes of protozoan parasites. Our understanding of CCs in cell signaling, organelle function, cellular homeostasis, and cell cycle control has led to improved insights into their structure and functions. In this article, we discuss CCs characteristics of five major protozoan parasites Plasmodium, Leishmania, Toxoplasma, Trypanosoma, and Cryptosporidium. We provide a comprehensive review of current antiparasitic drugs and the potential of using CCs as new therapeutic targets. Interestingly, previous studies have demonstrated that human CC modulators can kill or sensitize parasites to antiparasitic drugs. Still, none of the parasite CCs, pumps, or transporters has been validated as drug targets. Information for this review draws from extensive data mining of genome sequences, chemical library screenings, and drug design studies. Parasitic resistance to currently approved therapeutics is a serious and emerging threat to both disease control and management efforts. In this article, we suggest that the disruption of calcium homeostasis may be an effective approach to develop new anti-parasite drug candidates and reduce parasite resistance.

Ribosomal/nucleolar stress induction regulates tert-Butyl hydroperoxide (tBHP) mediated oxidative stress in Anopheles gambiae midguts.

OBJECTIVE: A fundamental understanding of redox homeostasis in Anopheles gambiae midgut cells under different oxidative conditions is missing. Such knowledge can aid in the development of new malaria transmission-blocking strategies aimed at disrupting natural homeostatic processes in the mosquito during Plasmodium parasite uptake (i.e. blood feeding). The aim of this study was to understand how the An. gambiae midgut regulates oxidative stress to reactive oxygen species (ROS), especially to a potent ROS-inducer such as tert-Butyl hydroperoxide (tBHP). RESULTS: Initial studies using quantitative immunoblot indicated that the expression of the classical antioxidant protein An. gambiae thioredoxin-1 (AgTrx-1) remained unchanged across challenges with different concentrations of tBHP suggesting that additional mechanisms to regulate ROS may be involved. We therefore conducted a global proteomic survey, which revealed that An. gambiae midguts under low (50 µM) and high (200 µM) tBHP concentrations were enriched in proteins indicative of ribosomal/nucleolar stress. Ribosomal stress is an inherent cellular response to an imbalance in ribosomal proteins (RPs) due to cellular stress such as oxidative stress. Our data suggest that ribosomal/nucleolar stress is the primary cellular response in An. gambiae midguts under tBHP challenge. Considering these results, we discuss harnessing the ribosomal stress response as a potential malaria transmission-blocking strategy.

Paraquat-Mediated Oxidative Stress in Anopheles gambiae Mosquitoes Is Regulated by An Endoplasmic Reticulum (ER) Stress Response.

Paraquat is a potent superoxide (O2-)-inducing agent that is capable of inducing an oxidative imbalance in the mosquito midgut. This oxidative imbalance can super-stress the malaria parasite, leading to arrested development in the mosquito midgut and reduced transmission. While several studies have explored the effect of paraquat on malaria parasites, a fundamental understanding of the mosquito response to this compound remains unknown. Here, we quantified the mosquito midgut proteomic response to a paraquat-laced sugar meal, and found that An. gambiae midguts were enriched in proteins that are indicative of cells under endoplasmic reticulum (ER) stress. We also carried out qRT-PCR analyses for nine prominent thioredoxin (Trx) and glutathione (GSH)-dependent genes in mosquito midguts post P. falciparum blood meal ingestion to evaluate the concordance between transcripts and proteins under different oxidative stress conditions. Our data revealed an absence of significant upregulation in the Trx and GSH-dependent genes following infected blood meal ingestion. These data suggest that the intrinsic tolerance of the mosquito midgut to paraquat-mediated oxidative stress is through an ER stress response. These data indicate that mosquitoes have at least two divergent pathways of managing the oxidative stress that is induced by exogenous compounds, and outline the potential application of paraquat-like drugs to act selectively against malaria parasite development in mosquito midguts, thereby blocking mosquito-to-human transmission.

Characterization of a protozoan Phosducin-like protein-3 (PhLP-3) reveals conserved redox activity.

We recently identified three novel thioredoxin-like genes in the genome of the protozoan parasite Plasmodium that belong to the Phosducin-like family of proteins (PhLP). PhLPs are small cytosolic proteins hypothesized to function in G-protein signaling and protein folding. Although PhLPs are highly conserved in eukaryotes from yeast to mammals, only a few representatives have been experimentally characterized to date. In addition, while PhLPs contain a thioredoxin domain, they lack a CXXC motif, a strong indicator for redox activity, and it is unclear whether members of the PhLP family are enzymatically active. Here, we describe PbPhLP-3 as the first phosducin-like protein of a protozoan organism, Plasmodium berghei. Initial transcription analysis revealed continuous low-level expression of pbphlp-3 throughout the complex Plasmodium life cycle. Attempts to knockout pbphlp-3 in P. berghei did not yield live parasites, suggesting an essential role for the gene in Plasmodium. We cloned, expressed and purified PbPhLP-3 and determined that the recombinant protein is redox active in vitro in a thioredoxin-coupled redox assay. It also has the capacity to reduce the organic compound tert-Butyl hydroperoxide (TBHP) in vitro, albeit at low efficiency. Sequence analysis, structural modeling, and site-directed mutagenesis revealed a conserved cysteine in the thioredoxin domain to be the redox active residue. Lastly, we provide evidence that recombinant human PhLP-3 exhibits redox activity similar to that of PbPhLP-3 and suggest that redox activity may be conserved in PhLP-3 homologs of other species. Our data provide new insight into the function of PhLP-3, which is hypothesized to act as co-chaperones in the folding and regulation of cytoskeletal proteins. We discuss the potential implications of PhLP-3 as a thioredoxin-target protein and possible links between the cellular redox network and the eukaryotic protein folding machinery.

Molecular cloning, characterization and expression profile of a glutathione peroxidase-like thioredoxin peroxidase (TPxGl) of the rodent malaria parasite Plasmodium berghei.

Glutathione peroxidases (GPx) comprise an important group of redox active proteins with diverse functions, including antioxidant defense and signaling. Although the genome of the malaria parasite Plasmodium does not contain a genuine GPx gene a glutathione peroxidase-like thioredoxin peroxidase (TPx(Gl)) has recently been identified and biochemically characterized in the human malaria parasite P. falciparum. To gain more insight into the potential biological function of this enzyme we have cloned and expressed TPx(Gl) of the rodent model system P. berghei (PbTPx(Gl)). Biochemical characterization confirmed that the protein is redox active with the P. berghei thioredoxin system. We compared PbTPx(Gl) to recently characterized thioredoxin-dependent GPx-type proteins of other organisms, and generated the first hypothetical 3D model of a Plasmodium TPx(Gl), which shows the conservation of the thioredoxin-fold as well as the spatial orientation of a classic GPx catalytic tetrad. In vivo studies indicate that PbTPx(Gl) is continuously expressed in all P. berghei asexual blood stages, gametocytes and in early mosquito-stage parasites. Confocal microscopy suggest a cytoplasmic localization of PbTPx(Gl) in all investigated parasite life stages, specifically in mature ookinetes. Our data provides new insights into the structure and ubiquitous expression of Plasmodium TPx(Gl) and warrants further investigation into this potentially important redox enzyme.

Multiple pathways for Plasmodium ookinete invasion of the mosquito midgut.

Plasmodium ookinete invasion of the mosquito midgut is a crucial step of the parasite life cycle but little is known about the molecular mechanisms involved. Previously, a phage display peptide library screen identified SM1, a peptide that binds to the mosquito midgut epithelium and inhibits ookinete invasion. SM1 was characterized as a mimotope of an ookinete surface enolase and SM1 presumably competes with enolase, the presumed ligand, for binding to a putative midgut receptor. Here we identify a mosquito midgut receptor that binds both SM1 and ookinete surface enolase, termed "enolase-binding protein" (EBP). Moreover, we determined that Plasmodium berghei parasites are heterogeneous for midgut invasion, as some parasite clones are strongly inhibited by SM1 whereas others are not. The SM1-sensitive parasites required the mosquito EBP receptor for midgut invasion whereas the SM1-resistant parasites invaded the mosquito midgut independently of EBP. These experiments provide evidence that Plasmodium ookinetes can invade the mosquito midgut by alternate pathways. Furthermore, another peptide from the original phage display screen, midgut peptide 2 (MP2), strongly inhibited midgut invasion by P. berghei (SM1-sensitive and SM1-resistant) and Plasmodium falciparum ookinetes, suggesting that MP2 binds to a separate, universal receptor for midgut invasion.

Transposon mutagenesis identifies genes essential for Plasmodium falciparum gametocytogenesis.

Gametocytes are essential for Plasmodium transmission, but little is known about the mechanisms that lead to their formation. Using piggyBac transposon-mediated insertional mutagenesis, we screened for parasites that no longer form mature gametocytes, which led to the isolation of 29 clones (insertional gametocyte-deficient mutants) that fail to form mature gametocytes. Additional analysis revealed 16 genes putatively responsible for the loss of gametocytogenesis, none of which has been previously implicated in gametocytogenesis. Transcriptional profiling and detection of an early stage gametocyte antigen determined that a subset of these mutants arrests development at stage I or in early stage II gametocytes, likely representing genes involved in gametocyte maturation. The remaining mutants seem to arrest before formation of stage I gametocytes and may represent genes involved in commitment to the gametocyte lineage.

Expression of cytosolic peroxiredoxins in Plasmodium berghei ookinetes is regulated by environmental factors in the mosquito bloodmeal.

The Plasmodium ookinete develops over several hours in the bloodmeal of its mosquito vector where it is exposed to exogenous stresses, including cytotoxic reactive oxygen species (ROS). How the parasite adapts to these challenging conditions is not well understood. We have systematically investigated the expression of three cytosolic antioxidant proteins, thioredoxin-1 (Trx-1), peroxiredoxin-1 (TPx-1), and 1-Cys peroxiredoxin (1-Cys Prx), in developing ookinetes of the rodent parasite Plasmodium berghei under various growth conditions. Transcriptional profiling showed that tpx-1 and 1-cys prx but not trx-1 are more strongly upregulated in ookinetes developing in the mosquito bloodmeal when compared to ookinetes growing under culture conditions. Confocal immunofluorescence imaging revealed comparable expression patterns on the corresponding proteins. 1-Cys Prx in particular exhibited strong expression in mosquito-derived ookinetes but was not detectable in cultured ookinetes. Furthermore, ookinetes growing in culture upregulated tpx-1 and 1-cys prx when challenged with exogenous ROS in a dose-dependent fashion. This suggests that environmental factors in the mosquito bloodmeal induce upregulation of cytosolic antioxidant proteins in Plasmodium ookinetes. We found that in a parasite line lacking TPx-1 (TPx-1KO), expression of 1-Cys Prx occurred significantly earlier in mosquito-derived TPx-1KO ookinetes when compared to wild type (WT) ookinetes. The protein was also readily detectable in cultured TPx-1KO ookinetes, indicating that 1-Cys Prx at least in part compensates for the loss of TPx-1 in vivo. We hypothesize that this dynamic expression of the cytosolic peroxiredoxins reflects the capacity of the developing Plasmodium ookinete to rapidly adapt to the changing conditions in the mosquito bloodmeal. This would significantly increase its chances of survival, maturation and subsequent escape. Our results also emphasize that environmental conditions must be taken into account when investigating Plasmodium-mosquito interactions.

The evolution and putative function of phosducin-like proteins in the malaria parasite Plasmodium.

Ubiquitous to the proteomes of all living species is the presence of proteins containing the thioredoxin (Trx)-domain. The best characterized Trx-domain containing proteins include the enzymes involved in cellular redox metabolism facilitated by their cysteine-containing active site. But not all members of the Trx-fold superfamily exhibit this catalytic motif, e.g., the phosducin-like (PhLP) family of proteins. Genome sequencing efforts have uncovered new Trx-domain containing proteins, and their redox activity and cellular functions have yet to be determined. The genome of the malaria parasite Plasmodium contains multiple thioredoxins and thioredoxin-like proteins which are of considerable interest given their role in the parasite's antioxidant defense. While adaptations within the Trx-domain have been studied, primarily with respect to redox active structures, PhLP proteins have not been examined. Using the uncharacterized phosducin-like protein from Plasmodium berghei PhLP-1, we investigated the evolution of PhLP proteins across all branches of the tree of life. As a result of our analysis, we have discovered the presence of two additional PhLP proteins in Plasmodium, PhLP-2 and PhLP-3. Sequence homology with annotated PhLP proteins in other species confirms that the Plasmodium PhLP-2 and PhLP-3 belong to the PhLP family of proteins. Furthermore, as a result of our analysis we hypothesize that the PhLP-2 thioredoxin was lost over time given its absence from higher-order eukaryotes. Probing deeper into the putative function of these proteins, inspection of the active sites indicate that PbPhLP-1 and PbPhLP-2 may be redox active while PbPhLP-3 is very likely not. The results of this phylogenetic study provide insight into the emergence of this family of Trx-domain containing proteins.

Stress response pathways in protozoan parasites.

Diseases caused by protozoan parasites have a dramatic impact on world health. Emerging drug resistance and a general lack of experimental understanding has created a void in the medicine cabinet used to treat these widespread infections. A novel therapeutic idea that is receiving more attention is centred on targeting the microbe's response to the multitude of environmental stresses it encounters. Protozoan pathogens have complex life cycles, often having to transition from one host to another, or survive in a cyst form in the environment until a new host arrives. The need to respond to environmental cues and stress, and endure in less than optimal conditions, is paramount to their viability and successful progression through their life cycle. This review summarizes the research on parasitic stress responses for Apicomplexa, kinetoplastids and anaerobic protozoa, with an eye towards how these processes may be exploited therapeutically.

Disruption of Plasmodium falciparum development by antibodies against a conserved mosquito midgut antigen.

Malaria parasites must undergo development within mosquitoes to be transmitted to a new host. Antivector transmission-blocking vaccines inhibit parasite development by preventing ookinete interaction with mosquito midgut ligands. Therefore, the discovery of novel midgut antigen targets is paramount. Jacalin (a lectin) inhibits ookinete attachment by masking glycan ligands on midgut epithelial surface glycoproteins. However, the identities of these midgut glycoproteins have remained unknown. Here we report on the molecular characterization of an Anopheles gambiae aminopeptidase N (AgAPN1) as the predominant jacalin target on the mosquito midgut luminal surface and provide evidence for its role in ookinete invasion. alpha-AgAPN1 IgG strongly inhibited both Plasmodium berghei and Plasmodium falciparum development in different mosquito species, implying that AgAPN1 has a conserved role in ookinete invasion of the midgut. Molecules targeting single midgut antigens seldom achieve complete abrogation of parasite development. However, the combined blocking activity of alpha-AgAPN1 IgG and an unrelated inhibitory peptide, SM1, against P. berghei was incomplete. We also found that SM1 can block only P. berghei, whereas alpha-AgAPN1 IgG can block both parasite species significantly. Therefore, we hypothesize that ookinetes can evade inhibition by two potent transmission-blocking molecules, presumably through the use of other ligands, and that this process further partitions murine from human parasite midgut invasion models. These results advance our understanding of malaria parasite-mosquito host interactions and guide in the design of transmission-blocking vaccines.

Entomopathogenic fungi as biological insecticides to control malaria.

Malaria is arguably the most serious vector-borne disease worldwide. The already-alarming number of deaths caused by malaria is increasing, caused in part by the increase in mosquito resistance to chemical insecticides. In two recent articles, the use of an approach was reported that could open a new front in the fight against malaria. Laboratory and field studies demonstrate that entomopathogenic fungi can efficiently kill adult anopheline mosquitoes, the females of which are the obligatory vectors for malaria parasites.

Immune signaling pathways regulating bacterial and malaria parasite infection of the mosquito Anopheles gambiae.

We show that, in the malaria vector Anopheles gambiae, expression of Cecropin 1 is regulated by REL2, an NF-kappaB-like transcription factor orthologous to Drosophila Relish. Through alternative splicing, REL2 produces a full-length (REL2-F) and a shorter (REL2-S) protein isoform lacking the inhibitory ankyrin repeats and death domain. RNA interference experiments show that, in contrast to Drosophila Relish, which responds solely to Gram-negative bacteria, the Anopheles REL2-F and REL2-S isoforms are involved in defense against the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli bacteria, respectively. REL2-F also regulates the intensity of mosquito infection with the malaria parasite, Plasmodium berghei. The adaptor IMD shares the same activities as REL2-F. Microarray analysis identified 10 additional genes regulated by REL2, including CEC3, GAM1, and LRIM1.

Origin of Toll-like receptor-mediated innate immunity.

Toll-related receptors (TLR) have been found in four animal phyla: Nematoda, Arthropoda, Echinodermata, and Chordata. No TLR has been identified thus far in acoelomates. TLR genes play a pivotal role in the innate immunity in both fruit fly and mammals. The prevailing view is that TLR-mediated immunity is ancient. The two pseudocoelomate TLRs, one each from Caenorhabditis elegans and Strongyloides stercoralis, were distinct from the coelomate ones. Further, the only TLR gene (Tol-1) in Ca. elegans did not appear to play a role in innate immunity. We argue that TLR-mediated innate immunity developed only in the coelomates, after they split from pseudocoelomates and acoelomates. We hypothesize that the function of TLR-mediated immunity is to prevent microbial infection in the body cavity present only in the coelomates. Phylogenetic analysis showed that almost all arthropod TLRs form a separate cluster from the mammalian counterparts. We further hypothesize that TLR-mediated immunity developed independently in the protostomia and deuterostomia coelomates.

The mosquito genome--a turning point?

Of the insects that serve as vectors for parasitic diseases, the genus Anopheles is the most important. Of the approximately 400 species, about twelve serve as vectors for human pathogens. Months have passed since the sequenced genomes of the malaria parasite, Plasmodium falciparum, and its vector, Anopheles gambiae, were published. Sequences were compared, gene and protein predictions were made, new research areas evolved and many ongoing projects gained new momentum. A general belief is that we are at a turning point: we are now in a position to tackle both the parasite and the vector from new angles and with new force, for example, by identifying new drug targets and obtaining a deeper insight into molecular mechanisms of the insect, the parasite and the interactions between them.

Plasmoredoxin, a novel redox-active protein unique for malarial parasites.

Thioredoxins are a group of small redox-active proteins involved in cellular redox regulatory processes as well as antioxidant defense. Thioredoxin, glutaredoxin, and tryparedoxin are members of the thioredoxin superfamily and share structural and functional characteristics. In the malarial parasite, Plasmodium falciparum, a functional thioredoxin and glutathione system have been demonstrated and are considered to be attractive targets for antimalarial drug development. Here we describe the identification and characterization of a novel 22 kDa redox-active protein in P. falciparum. As demonstrated by in silico sequence analyses, the protein, named plasmoredoxin (Plrx), is highly conserved but found exclusively in malarial parasites. It is a member of the thioredoxin superfamily but clusters separately from other members in a phylogenetic tree. We amplified the gene from a gametocyte cDNA library and overexpressed it in E. coli. The purified gene product can be reduced by glutathione but much faster by dithiols like thioredoxin, glutaredoxin, trypanothione and tryparedoxin. Reduced Plrx is active in an insulin-reduction assay and reduces glutathione disulfide with a rate constant of 640 m-1.s-1 at pH 6.9 and 25 degrees C; glutathione-dependent reduction of H2O2 and hydroxyethyl disulfide by Plrx is negligible. Furthermore, plasmoredoxin provides electrons for ribonucleotide reductase, the enzyme catalyzing the first step of DNA synthesis. As demonstrated by Western blotting, the protein is present in blood-stage forms of malarial parasites. Based on these results, plasmoredoxin offers the opportunity to improve diagnostic tools based on PCR or immunological reactions. It may also represent a specific target for antimalarial drug development and is of phylogenetic interest.

Immunity-related genes and gene families in Anopheles gambiae.

We have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster. Immune-related gene families involved in recognition, signal modulation, and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different life-styles. In contrast, the multifunctional Toll signal transduction pathway is substantially conserved, presumably because of counterselection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to expansion of the immune repertoire.

Thioredoxin-2 but not thioredoxin-1 is a substrate of thioredoxin peroxidase-1 from Drosophila melanogaster: isolation and characterization of a second thioredoxin in D. Melanogaster and evidence for distinct biological functions of Trx-1 and Trx-2.

As Drosophila melanogaster does not contain glutathione reductase, the thioredoxin system has a key function for glutathione disulfide reduction in insects (Kanzok, S. M., Fechner, A., Bauer, H., Ulschmid, J. K., Müller, H. M., Botella-Munoz, J., Schneuwly, S., Schirmer, R. H., and Becker, K. (2001) Science 291, 643-646). In view of these unique conditions, the protein systems participating in peroxide metabolism and in redox signaling are of special interest. The genes for a second thioredoxin (DmTrx-2) and a thioredoxin peroxidase (DmTPx-1) were cloned and expressed, and the proteins were characterized. In its disulfide form, the 13-kDa protein thioredoxin-2 is a substrate of thioredoxin reductase-1 (K(m) = 5.2 microm, k(cat) = 14.5 s(-1)) and in its dithiol form, an electron donor for TPx-1 (K(m) = 9 microm, k(cat) = 5.4 s(-1)). DmTrx-2 is capable of reducing glutathione disulfide with a second order rate constant of 170 m(-1) s(-1) at pH 7.4 and 25 degrees C. Western blot analysis indicated that this thioredoxin represents up to 1% of the extractable protein of D. melanogaster Schneider cells or whole fruit flies. Recombinant thioredoxin peroxidase-1 (subunit molecular mass = 23 kDa) was found to be a decameric protein that can efficiently use Trx-2 but not Trx-1 as a reducing substrate. The new electron pathway found in D. melanogaster is also representative for insects that serve as vectors of disease. As a first step we have cloned and functionally expressed the gene that is the orthologue of DmTrx-2 in the malaria mosquito Anopheles gambiae.